MICRODROPS AND FLOW SORTING FOR CHROMOSOME ISOLATION

流式分选是一种分离染色体的强大技术。 纯染色体片段对人类基因组计划至关重要。 纯化染色体亚片段和某些染色体（特别是 鼠标）是不可能的，然而，由于目前的限制， 技术. 该项目提供了解决这些限制的方法 通过开发用于序列特异性荧光标记的方法， 用DNA探针对染色体进行流式分选， 标记的染色体。 这一建议代表了 建立一种简单的方法，用于物理分离特定的大型 染色体片段（大于或等于1兆碱基）的数量 适合做实验。 单个染色体将被封装到凝胶微滴（GMD）中。 GMD染色体单元是一个物理上可操作的测试实体。 在 使用对所需染色体特异的DNA探针的原位杂交将 在GMD染色体单元内进行。 在特定标记之后， 将通过流式分选分离GMD包封的染色体。 这 这种方法结合了原位杂交的特异性和 自动化的分离技术。 的具体目标 （一）建立原位杂交技术， （二）制定封装单个GMD的程序， GMD中的染色体，（iii）证明染色体的特异性标记 在GMD内，以及（iv）分离特异性标记的GMD-染色体单位 通过流式分选。

Flow sorting is a powerful technology for the isolation of chromosomes. Pure chromosome fractions have been essential to the human genome project. Purification of chromosome sub-fragments and some chromosomes (especially mouse) is not possible, however, due to limitations in the current technology. This project provides a means for addressing these limitations by developing methods for sequence-specific fluorescent labelling of chromosomes with DNA probes followed by flow sorting of specifically labelled chromosomes. This proposal represents the first step in the development of a simple method for the physical isolation of specific large chromosomal fragments (greater than or equal to 1 megabase) in amounts suitable for experimentation. Individual chromosomes will be encapsulated into gel microdrops (GMDs). The GMD-chromosome unit is a physically manipulable testing entity. In situ hybridization using DNA probes specific to the desired chromosome will be performed within the GMD-chromosome unit. Following specific tagging, the GMD-encapsulated chromosomes will be isolated by flow sorting. This approach combines the specificity of in situ hybridization with the power of automated separation by flow sorting. The specific goals of the research are: (i) To develop procedures for in situ hybridization within GMDs, (ii) To develop procedures for encapsulation of individual chromosomes in GMDs, (iii) To demonstrate specific labelling of chromosomes within GMDs, and (iv) To isolate specifically labeled GMD-chromosome units by flow sorting.